The present invention relates generally to the area of bioinformatics and more specifically to methods, algorithms and apparatus for computer directed polynucleotide assembly. The invention further relates to the production of polypeptides encoded by polynucleotides assembled by the invention.
Enzymes, antibodies, receptors and ligands are polypeptides that have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism. The use of a polypeptide for specific technological applications may require the polypeptide to function in environments or on substrates for which it was not evolutionarily selected. Polypeptides isolated from microorganisms that thrive in extreme environments provide ample evidence that these molecules are, in general, malleable with regard to structure and function. However, the process for isolating a polypeptide from its native environment is expensive and time consuming. Thus, new methods for synthetically evolving genetic material encoding a polypeptide possessing a desired activity are needed.
There are two ways to obtain genetic material for genetic engineering manipulations: (1) isolation and purification of a polynucleotide in the form of DNA or RNA from natural sources or (2) the synthesis of a polynucleotide using various chemical-enzymatic approaches. The former approach is limited to naturally-occurring sequences that do not easily lend themselves to specific modification. The latter approach is much more complicated and labor-intensive. However, the chemical-enzymatic approach has many attractive features including the possibility of preparing, without any significant limitations, any desirable polynucleotide sequence.
Two general methods currently exist for the synthetic assembly of oligonucleotides into long polynucleotide fragments. First, oligonucleotides covering the entire sequence to be synthesized are first allowed to anneal, and then the nicks are repaired with ligase. The fragment is then cloned directly, or cloned after amplification by the polymerase chain reaction (PCR). The polynucleotide is subsequently used for in vitro assembly into longer sequences. The second general method for gene synthesis utilizes polymerase to fill in single-stranded gaps in the annealed pairs of oligonucleotides. After the polymerase reaction, single-stranded regions of oligonucleotides become double-stranded, and after digestion with restriction endonuclease, can be cloned directly or used for further assembly of longer sequences by ligating different double-stranded fragments. Typically, subsequent to the polymerase reaction, each segment must be cloned which significantly delays the synthesis of long DNA fragments and greatly decreases the efficiency of this approach.
The creation of entirely novel polynucleotides, or the substantial modification of existing polynucleotides, is extremely time consuming, expensive, requires complex and multiple steps, and in some cases is impossible. Therefore, there exists a great need for an efficient means to assemble synthetic polynucleotides of any desired sequence. Such a method could be universally applied. For example, the method could be used to efficiently make an array of polynucleotides having specific substitutions in a known sequence that is expressed and screened for improved function. The present invention satisfies these needs by providing efficient and powerful methods and compositions for the synthesis of a target polynucleotide encoding a target polypeptide.
The present invention provides methods for the synthetic assembly of polynucleotides and related algorithms. In particular, the present invention provides fast and efficient methods for generating any nucleic acid sequence, including entire genes, chromosomal segments, chromosomes and genomes. Because this approach is based on a completely synthetic approach, there are no limitations, such as the availability of existing nucleic acids, to hinder the construction of even very large segments of nucleic acid.